Cross-Transfusion of Postshock Mesenteric Lymph Provokes Acute Lung Injury

Cross-transfusion of Postshock Mesenteric Lymph ProvokesAcute Lung Injury

M. Wohlauer1, E. Moore1,2, J. Harr1, J. Eun1, M. Fragoso1, A. Banerjee2, and CC Silliman3,4

1Department of Surgery, University of Colorado Denver, Denver, CO.2Department of Surgery, Denver Health Medical Center, Denver, CO.3Department of Pediatrics, University of Colorado Denver, Denver, CO.4Research Department, Bonfils Blood Center, Denver, CO.

AbstractObjective—Substantial investigation has implicated mesenteric lymph as the mechanistic linkbetween gut ischemia/reperfusion (I/R) and distant organ injury. Specifically, lymph diversionprevents acute lung injury (ALI) in vitro and bioactive lipids and proteins isolated from postshockmesenteric lymph (PSML), maintain bioactivity in vitro. However, Koch’s postulates remain to besatisfied via direct cross-transfusion into a naïve animal. We therefore hypothesized that real timecross-transfusion of postshock mesenteric lymph provokes acute lung injury.

Methods—One set of Sprague-Dawley rats (lymph donors) was anesthetized, with themesenteric lymph ducts cannulated and exteriorized to drain freely into a siliconized plastic cup;concurrently, a second group of rats (lymph recipients) was anesthetized, with a cannula insertedinto the animal’s right internal jugular vein. Blood was removed from the donor rats to inducehemorrhagic shock (MAP of 35 mmHg × 45 minutes). The recipient rats were positioned 10 cmbelow the plastic cup which emptied into the jugular vein cannula. Thus, mesenteric lymph fromthe shocked donor rat was delivered to the recipient rat at the rate generated during shock and thesubsequent 3 hours of resuscitation.

Results—Neutrophil (PMN) accumulation in the lungs was substantially elevated in thepostshock lymph cross-transfusion group compared to both sham lymph cross-transfusion andinstrumented control (MPO: 9.42±1.55 vs. 2.81±0.82 U/mg lung tissue in postshock vs. shamlymph cross-transfusion, n=6 in each group, p=0.02). Additionally, cross-transfusion of PSMLinduced oxidative stress in the lung (0.21±0.03 vs. 0.10±0.01 micromoles MDA per mg lungtissue in lymph cross-transfusion vs. instrumented control, n=6 in each group, p = 0.046).Furthermore, transfusion of PSML provoked lung injury (BAL protein 0.77±0.18 vs. 0.15±0.02mg/ml protein in BALF, postshock vs. sham lymph cross-transfusion, n=6 in each group,p=0.004).

Conclusion—Cross-transfusion of PSML into a naïve animal leads to PMN accumulation andprovokes ALI. These data provide evidence that postshock agents released into mesenteric lymphare capable of provoking distant organ injury.

© 2011 Elsevier Inc. All rights reserved.Corresponding Author: Ernest E. Moore, MD, Phone: 303-436-6561 Fax: 303-436-6572 [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptJ Surg Res. Author manuscript; available in PMC 2012 October 1.

Published in final edited form as:J Surg Res. 2011 October ; 170(2): 314–318. doi:10.1016/j.jss.2011.03.045.

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INTRODUCTIONMultiple organ failure (MOF), the leading cause of late mortality and a major source ofmorbidity in trauma patients, is the net result of a maladaptive systemic inflammatoryresponse to injury. Despite intensive investigation, the supportive care initially proposed byEiseman nearly 35 years ago remains the standard treatment for MOF, and there is noeffective pharmacologic therapy for this devastating illness.1 Acute lung injury (ALI) is thefirst clinical manifestation of organ failure, followed by hepatic and renal dysfunction.2Although animal studies initially implicated gut bacterial translocation, a prospectiveclinical study was unable to detect endotoxin in the portal circulation of trauma patientsdespite a 30% incidence of MOF in this population.3 It is now understood that circulatoryshock is integral in the early pathogenesis of MOF. Postshock mesenteric lymph (PSML)has been implicated as the conduit by which bioactive exudates from the ischemic gut aredelivered into the circulation. 4,5 This is supported by the fact that lymphatic diversion priorto trauma/hemorrhagic shock (T/HS) prevents remote organ injury.6,7 Tissue injury andhypoperfusion unleash a number of products which appear in the lymph: cytokines,8inflammatory lipids, coagulation factors,9 and cellular breakdown products.10 The lymphaticsystem collects the extravasated fluid and cellular breakdown products, proteins, and lipidsfrom the interstitial space and returns them to the blood circulation via the subclavian vein.The protein composition of lymph is nearly identical to that of interstitial fluid, and thoughtto be similar to, although less concentrated, than that of plasma.11 An important exception isthe gut, which has the ability to acquire factors from outside of the cardiovascular systemand deliver them to the circulation via a mesenteric lymph conduit, giving the injured gut aunique descriptor: the “motor of MOF.12

In 1882, Robert Koch published a landmark paper describing microbial pathogenicity. Hismethods of linking a specific bacterium to a disease, termed Koch’s postulates, are soundprinciples applicable to a variety of fields in translational research: 1) the microorganism ispresent and discoverable in every case of the disease; 2) the microorganism is to becultivated in a pure culture; 3) inoculations from such culture must reproduce the disease insusceptible animals; 4) it must be re-obtained from such animals and again grown in a pureculture.13 After having satisfied Koch’s first 2 postulates in previous work, we sought tosatisfy Koch’s 3rd postulate – hypothesizing that real time cross-transfusion of postshockmesenteric lymph (PSML) into a naïve animal will provoke acute lung injury.

METHODSTrauma/Hemorrhagic Shock Model

Sprague-Dawley rats weighing 350g to 400g (Harlan Labs, Indianapolis, IN) were housed ina climate-controlled barrier facility with 12 hr light/dark cycles and free access to food andwater for a period of at least one week prior to experimental procedures. General anesthesiawas performed with an intraperitoneal injection of 50 mg/kg sodium pentobarbital, and localanesthesia with a subcutaneous injection of 1% lidocaine. Body temperature was monitoredrectally and euthermia maintained with a heat lamp. The femoral artery and vein werecannulated with PE 50 tubing and blood pressure monitored using a ProPaq invasivemonitoring device (Welch Allyn Inc., Skaneateles Falls, NY). A separate skin incision wascreated to tunnel the femoral catheters prior to closure of the groin incision. A 3 cm midlinelaparotomy was performed, simulating tissue injury with trauma. The bowel was evisceratedand rotated to the left to expose the mesenteric duct and accessory duct (located adjacent tothe superior mesenteric artery) which were isolated by blunt dissection. The main lymphaticduct was cannulated with PE 100 tubing, secured with 7-0 prolene suture, and maintained inplace with Super Glue (Rancho Cucamonga, CA). The tubing was then exteriorized via aright flank stab wound, and the accessory lymph duct was then ligated. The laparotomy

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incision was closed in a two-layer fashion, and the mesenteric lymph transfused directly intothe right internal jugular vein of the anesthetized naïve rat (Figure 1). In the donor rat, shockwas induced by controlled hemorrhage to a MAP of 35 mmHg which was sustained for 45min by the withdrawal or return of shed blood (SB). Hypotension was maintained until thebase deficit was >20 meq/L.

Resuscitation of the donor rat was performed by infusing twice the SB volume in NS over30 min, half the volume of the shed blood over 30 min, and then twice the SB volume in NSover 60 min via the femoral vein. Lymph cross-transfusion continued for 3 hrs (parallelingthe 2 hr resuscitation period followed by the 1 hr observation period in the hemorrhagicshock or sham shock donor rat). A bronchoalveolar lavage was performed in the recipientrat; both animals were euthanized via a pentobarbital overdose. In the sham lymph cross-transfusion group, the donor rat received a laparotomy without hemorrhagic shock, withtransfusion of sham lymph for 3 hours. In the instrumented control group, the internaljugular vein was cannulated with infusion of normal saline, at an equivalent volume and rate(3 ml/hr). Plasma and BAL samples were centrifuged at 400g at 4° C for 10 minutes, andsnap frozen in liquid nitrogen and stored at −80° C.

Tissue and BAL Fluid AnalysisA shock state leads to sequestration of large numbers of polymorphonuclear leukocytes inthe lungs.14 When provoked, these neutrophils provoke acute lung injury via release of toxicmetabolites.15 Therefore, lung PMN accumulation was evaluated using the myeloperoxidase(MPO) assay. Lung tissue stored at −80° C, was thawed, weighed, and homogenized in 10mL of 20 mmol/L potassium phosphate buffer (PPB) and centrifuged at 40,000g for 30minutes. The pellet was sonicated for 90 seconds in 4 mL of 50 mmol/L PPB containing 0.5g/dL hexadecyltrimethyl ammonium bromide, incubated at 60 C and centrifuged. Thesupernatant (5 µL) was added to 145 µL of 50 mmol/L PPB containing 0.167 mg/mL O-dianisidine with 0.0005% hydrogen peroxide; the absorbance at 460 nm was measured witha spectrophotometer (Molecular Devices Corp, Sunnyvale, Calif) to determine MPOactivity.16 Oxygen metabolites generated by PMNs in the lung have been shown to provokeacute lung injury.17 Therefore, malondialdehyde (MDA), a product of lipid peroxidation,was used to measure oxidative stress in the lung tissue (TBARS assay kit, Cell Biolabs, Inc.,San Diego, CA) using a colorimetric assay.

BAL protein was used as a direct marker of lung damage. Normal saline (5 ml) was injectedinto the trachea (three times) and collected, with the return of bronchoalveolar lavage fluid(BALF) consistently greater than 12 mL. The BALF was then centrifuged at 400g at 4° Cfor 10 minutes and protein quantification performed using the BCA protein analysis methodwith bovine serum albumin standards. Data are reported as mean ± SEM. Student’s t-testwas used to determine statistical significance.

RESULTSLung neutrophil accumulation following lymph cross-transfusion

Neutrophils, critical in the pathogenesis of ALI, were significantly elevated following lymphcross-transfusion. PMN accumulation in the lungs as measured by myeloperoxidase (MPO)activity was substantially higher in the lymph cross-transfusion group is shown in Figure 2:(MPO: 9.42±1.55 vs. 2.81±0.82 U/mg lung tissue in postshock vs. sham lymph cross-transfusion), n=6 in each group, p=0.02). The sham lymph cross-transfusion group levelswere comparable to the instrumented controls (MPO: 2.81±0.82 vs. 2.47±0.02 U/mg lungtissue, p=0.35).

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Cross-transfusion induces PMN-generated oxidative stress in the lungMalondialdehyde (MDA), a product of lipid peroxidation, was used to measure oxidativestress in the lung tissue following cross-transfusion of PSML into a naïve rat. Transfusion ofpostshock mesenteric lymph induced oxidative stress in the lung (Figure 3: 0.21±0.03 vs.0.10±0.01 micromoles MDA per mg lung tissue in lymph cross-transfusion vs. sham, p =0.046).

Postshock lymph transfusion provokes an increase in lung vascular permeabilityThe primary study endpoint was ALI, determined by protein extravasation into the alveolarspace at 3 hours postinjury. As illustrated in Figure 4, transfusion of mesenteric lymphprovoked substantial acute lung injury (BAL protein 0.77±0.18 vs. 0.15±0.02 mg/ml proteinin BALF, postshock lymph vs. sham lymph cross-transfusion, n=6 in each group, p=0.004).The sham lymph cross-transfusion group levels were comparable to the instrumentedcontrols (0.15±0.02 vs. 0.14±0.06 mg/ml, p=0.38).

DISCUSSIONRobert Koch was the first person to demonstrate a strict causative relationship between amicroorganism and disease. Before his pioneering work, the diagnosis of tuberculosis was amatter of judgment and dispute, described by a group of syndromes: miliary tuberculosis,caseous bronchitis, phthisis, scrofula, and bovine tuberculosis, with no universal agreementas to whether these were manifestations of the same condition. Since that time, Koch’spostulates have been applied to a variety of research fields, including atherosclerosis,18

microbial genetics,19 and the gut lymph hypothesis.20

Acute lung injury, ARDS, and multiple organ failure are a group of syndromes caused bysystemic hyperinflammation, in which morbidity is high and treatment remains supportive.The combined insults of trauma and hemorrhagic shock lead to the release of toxicinflammatory products from the gut into the circulation. Initially, loss of gut barrier functionand bacterial translocation was the predominant explanation for the development of MOF.12

In a prospective clinical study exploring this hypothesis, however, our group did not findendotoxin in the portal circulation of trauma patients with MOF.3 Subsequent clinicalstudies have supported this finding.21,22 After the bacterial translocation mechanism failedto satisfy Koch’s postulates, the lymphatic system became the target of investigation as theprimary conduit for delivering inflammatory factors from the ischemic gut into thecirculation.

Koch’s postulates (adapted to the gut lymph hypothesis):

1. The inflammatory mediator must be found in all organisms with the disease, and isnot present in animals without the disease. A factor (or factors) in the PSMLprovokes acute lung injury following hemorrhagic shock, and in the absence ofPSML due to ligation of the mesenteric duct, acute lung injury does not occur. 6,7

2. The inflammatory mediator must be isolated from a diseased organism andmaintain activity in vitro. PSML provokes PMN priming 23,24 and is toxic to humanendothelial cells in vitro.25,26

3. The inflammatory mediator should cause disease when introduced into a healthyorganism. Injection of PSML provokes acute lung injury when transfused into anaïve animal.

4. The inflammatory mediator must be re-isolated from the inoculated, diseasedexperimental host and identified as being identical to the original specific causative

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agent. With Koch’s 3rd postulate satisfied, future experiments are designed to testKoch’s 4th and final postulate.

Infusion of stored PSML into naïve rats has been shown to provoke ALI;27 however, lymphcontains volatile peptides and lipids, sensitive to storage, freeze/thaw cycles, includingclotting factors prone to contact activation. Concerned that processing the lymph may havealtered its bioactivity, a method was developed to deliver PSML directly from themesenteric duct from a T/HS rat into the jugular vein of a naïve rat, which provoked ALI.Real time transfusion of sham lymph resulted in lung injury similar to instrumented controlsgiven normal saline only. This observation supports previous work that preshock lymphdoes not cause inflammation, and may even be protective against ALI.28

Because the lymphatic system is highly permeable, there is little, if any, exclusion ofinterstitial molecules.11 Mesenteric lymph contains a number of inflammatory productsincluding arachidonic acid,29 serine proteases,30 cytokines, 8,31 cellular breakdownproducts, 32,33 coagulation factors,9,10 and modified albumin.34 Bacteria and bacterialproducts are noticeably absent in PSML.35 Additionally, PSML demonstrates increasedlevels of anti-inflammatory cytokines21 and depletion of protease inhibitors.10

Phospholipase A2 (PLA2), an enzyme abundant in the mammalian gut has been implicatedas the source of lipid mediators in the setting of hemorrhagic shock and sepsis.36 PLA2produces arachidonic acid, a precursor to a variety of inflammatory products found in thelymph, leukotrienes, thromboxane, and prostaglandins. Arachidonic acid is abundant inPSML, and leukotrienes are increased in the lungs of animals following T/HS, making theselipids the current focus of intensive investigation in our lab.37 Postshock mesenteric lymphmay be toxic through a variety of mechanisms, including endothelial inflammation,6,26

neutrophil priming,38,39,40 enhanced apoptosis,41 pulmonary epithelial inflammation,42 andRBC hemolysis.43,32

In this study, we demonstrate that postshock mesenteric lymph is necessary and sufficient toprovoke ALI in the naïve rat. These data further support a central mechanistic role of PSMLin the pathogenesis of ALI. Although the postshock lung may also play a role, the cross-transfusion model will facilitate mechanistic investigation, compartmentalizing the gut-lymph versus ischemic lung contributions.

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[PubMed: 841449]2. Ciesla DJ, Moore EE, Johnson JL, et al. The role of the lung in postinjury multiple organ failure.

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perspective with major torso trauma. J Trauma. 1991; 31:629–636. [PubMed: 2030509]4. Moore EE, Moore FA, Franciose RJ, et al. The postischemic gut serves as a priming bed for

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Figure 1.The method of real-time cross-transfusion of mesenteric lymph from a donor rat undergoingtrauma and hemorrhagic shock to a naïve recipient is illustrated.

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Figure 2.PMN accumulation in the lungs was substantially elevated in the postshock lymph cross-transfused group (9.42±1.55 vs. 2.81±0.82 U/mg lung tissue in postshock vs. sham lymphcross-transfusion, indicated by an asterisk, p=0.02). The sham lymph cross-transfusiongroup levels were comparable to the instrumented controls (2.81±0.82 vs. 2.47±0.02 U/mglung tissue, indicated by a number sign, p=0.35).

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Figure 3.Oxidative lung injury was increased in the lymph transfused animals (MDA 0.21 ± 0.03 vs.0.10 ± 0.01 micromoles MDA per mg lung tissue in lymph cross-transfusion vs.instrumented control, p = 0.046)

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Figure 4.Transfusion of mesenteric lymph provoked acute lung injury (BAL protein 0.77±0.18 vs.0.15±0.02 vs. 0.14±0.06 mg/ml protein in BALF, postshock lymph vs. sham lymph cross-transfusion, indicated by an asterisk, p=0.004). The sham lymph cross-transfusion grouplevels were comparable to the instrumented controls (0.15±0.02 vs. 0.14±0.06 mg/ml,indicated by a number sign, p=0.38).

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Cross-Transfusion of Postshock Mesenteric Lymph Provokes Acute Lung Injury

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